This invention relates to immunohistochemistry, and in particular relates to a spray nozzle for apparatus for automatically staining tissue sections, or cell preparations.
Histochemistry is a branch of biochemistry devoted to the study of the chemical composition and structure of animal and plant tissues. It involves the use of microscopic, x-ray diffraction and radioactive tracer techniques in examining the cellular composition and structure of bones, blood, muscle and other animal and vegetable tissues.
Immunohistochemistry is useful for directly viewing the cellular distribution of a molecule (marker) using labelled antibodies or other ligands including nucleic acid probes. Labels include enzymes, radioisotopes and fluorescent molecules. The technique can be applied to whole cells, for example for identification of lymphomas (white blood cell cancers) or to tissue sections, for example for cancer diagnosis.
The specific (or primary) antibody may be labelled directly. Alternatively and more often, a second antibody carrying the label is used to specifically bind to the first. Also, the tissue may require pretreatment to reveal the marker of interest (enzymic) or to remove non-specific effects.
A number of methods have been developed to amplify the visual signal and this may add several steps to the technique. Ultimately, in the case of an enzyme-label a substrate is applied which produces a coloured product at the site of the label. The surrounding tissue is then counterstained to provide contrast. Therefore most protocols for carrying out staining involve a large number of incubations of various time periods, separated by washing to remove spent reagents.
Each operation requires considerable care in the application of small amounts (50 to 200 microliters) of reagents to cover the tissue adequately and also in the washing steps to ensure complete removal of spent reagents and to avoid accidental removal of tissue from the slide. Thus, when a large number of slides are involved the procedure is labour intensive, tedious and can suffer from a lack of reproducibility.
A manual technique currently utilised for staining tissue involves a skilled technician performing all operations manually.
Firstly, glass specimen slides are supported on trays. When staining is carried out above ambient temperature, heating is normally provided using a specially designed temperature-controlled template with provision to allow high humidity.
The slides are washed with a buffer stream from a hand-operated dispensing bottle. The slides are then cleared of liquid by being set vertically to drain, and wiped around the specimen with paper towel material.
The biochemical agent delivery is via a manual pipettor positioned by eye such that the fluid is spread to cover the tissue sample. Chemical reagents with short active lives are manually mixed in vials using standard pipettors.
The control of event sequences and times is performed manually with the aid of a stopwatch and note pads.
It is obvious that such a manual process is inherently inaccurate, time consuming and costly.
There have been prior attempts to automate such a process, and details of such attempts appear hereunder.
(1) Stross, W. P., Jones, M., Mason, D. Y. J. Clin. Pathol. January 1989 42(1) p 106-112 These authors have modified en existing tissue processing instrument (Histokinette E7326; British American Optical Corporation) to carry out immunohistochemical staining of tissues in a semi-automated manner. Slides are placed in racks which are dipped into tanks of reagents which are used repeatedly for up to four months. The method was only applied to the (APAAP) Alkaline phophatase staining method and did not automate the application of primary antibody or substrate. PA1 (2) Brigati, D. J., Budgeon, L. R., Unger, E. R., Koebler, D., Cuomo, C., Kennedy, T., Perdomo, J. M. J. Histotechnol. 11(3), 1988 p 165-183 These authors claim to have developed the first automated method for immunocytochemistry. The method uses a triaxial robotic slide system to move racks of slides between reagents. The slides are paired so that reagents fill the gaps between slides by capillary action. The system is claimed to be able to carry out the complete immunostaining procedure as well as in-situ hybridisation and has been commercialised by Fischer Scientific Co. (U.S.A.). PA1 (3) Stark, E., Faltinat, D., Von der Fecht, R., J. Immunological Methods 107 (1988) p 89-92 These authors have described an instrument in which up to 30 slides are carried on a carousel which can be rotated rapidly to remove reagents. The antibodies or other solutions are piperted onto the slides by standard plastic syringes. This approach has a major disadvantage in that it uses large volumes of expensive reagents and does not automate the primary antibody step. PA1 (4) Mehven, L., Med. Lab. World February 1989 p 45-46 Described a novel coverslip device which is used to create a capillary gap between the slide and coverslip. Reagents are transfered from vials in a carousel to a funnel part of the coverslip using an automatic pipette and an x-slide device. 10 basic method programs can be used to run 20 slides automatically with a variety (up to 9) primary antibodies. The instrument has been commercialised by Shandon Scientific (U.K.) under the "Cadenza" trade name. PA1 (5) Recently Lipshaw (U.S.A.) have released an instrument for the automatic staining of batches of slides by the peroxide technique. The system involves the transfer of a rack of slides between baths of reagents with a robotic arm. The instrument is limited to use for slides undergoing identical methods, is not useful for adding primary antibody and uses large volumes of other reagents.